TEMPLATE DESIGN © Eliminating non-specific interactions for accurate single- molecule force measurements with magnetic tweezers Noah Johnson †,‡, Gokul Upadhyayula †, Dr. Sharad Gupta †, Dr. Valentine Vullev † † University of California, Riverside, CA 92521, ‡ The Pennsylvania State University, University Park, PA Abstract Magnetic tweezers (MT) are widely used for investigating nanometer-sized molecular complex interactions by applying forces to micrometer-sized superparamagnetic beads. In contrast to other force measurement techniques such as optical tweezers and atomic force microscopy, magnetic tweezers offer a few key advantages: (1) MT allow for the recording of hundreds of single-molecule events in parallel with a single measurement; (2) Magnetic forces are orthogonal to most biological interactions which eliminates the risk of altering sample properties during MT measurements; (3) MT require relatively low energy, significantly reducing the risk of sample overheating; (4) MT are able to conduct measurements at a constant force, eliminating the need to take loading rates into consideration. Due to significant measurement error caused by non-specific interactions between probe interfaces at nanometer separation, the utilization of single-molecule force measurements remains largely unexplored. By employing surface-engineering methodologies developed in our laboratory, we aim to suppress the non-specific interactions between the slide surface and the beads, greatly increasing the accuracy of our measurements. Once both surfaces are derivatized, we will use MT for dissociation studies of protein- ligand complexes, and furthermore to study the directionality of dissociation by strategically attaching the bead and substrate at various positions. Force Calibration Using Stokes equation we can calculate the drag force. The gravitational force can be calculated theoretically. Calibration Results We controlled the distance between the magnet and the center of the field of view as well as the electromagnet voltage. We compiled a calibration matrix with distances between 1 and 5mm and magnet voltages between 2 and 12V. Figure 2 – The calibration was performed with 3μm diameter polypropylene beads in a 1.33mM TWEEN 20 surfactant solution, used to keep the beads from sticking together. Eliminating Non-specific Interactions To accurately study protein-ligand dissociation kinetics we must minimize the non-specific interactions between the beads and the substrate. The effects of Van der Waals forces are drastically reduced by separating beads and surfaces (Figure 3). MT is an inverted microscope with a magnet. However, in the calibration setup, the objective is on the side to observe the movement of beads in the presence of an external magnetic field (Figure 1). Through this setup, we can calculate the terminal velocity of beads at specific voltages. N CCD Figure 1 1mm square glass capillary with bead suspension 10x objective 12V electromagnet CCD camera S Once bead velocity is calculated, we can assume the magnetic force on the bead is equal to the drag force of suspension fluid on the bead plus the gravitational force on the bead. FgFg FMFM FdFd F M = F d +F g F d =6π η rv η = Fluid viscosity r = Bead radius v = Bead velocity F g = mg = ρ beads V fluid g Thus far we have observed forces ranging from to 2.44pN. distance Figure 3 glass silanization deprotection reductive amination glass 11 nm Figure 4 1 Figure 5 Each PEG layer adds ~11nm of separation to the surface. Entropic repulsion by PEGylation reduces Van der Waals forces by nearly five orders of magnitude (Figure 3). References Acknowledgements Results The surfaces and beads were successfully derivatized with PEG 3000, resulting in a contact angle of 72.4±0.79° for the coated glass surface compared to 26.7±0.24°. We found the 1.33mM TWEEN 20 to work very well in keeping the beads separated. A B Figure 6 – (A) Significant bead clumping is seen at 40x in distilled water. (B) Single beads are seen in TWEEN 20 suspension solution at 10x. 1)Wan, J., Thomas, M., Guthrie, S., & Vullev, V., “Surface-Bound Proteins with Preserved Functionality,” Annals of Biomed Eng, 2009, 6, We would like to thank Sean Guthrie who helped with derivatization procedures and Stephen Bishop who helped take calibration data. Also Jun Wang for organizing the 2009 UCR BRITE REU program, and the National Science Foundation for funding. We applied a tiny force of 0.357pN with the MT (Figure 7) for 30s to test the effectiveness of PEGylation in minimizing non-specific interactions. B A Using surface engineering, we derivatized the glass and beads with PEG (Figures 4 & 5 respectively). Figure 7 – (A) Non-PEGylated beads and surface before and after the force is applied. (B) PEGylated beads and surface.